MCL1 is an antiapoptotic BCL2 family member that acts at a pivotal point in the apoptotic cascade and that is dynamically regulated to control viability at critical stages in cell proliferation and differentiation. Alterations that affect its normal regulated expression pattern can contribute to cancer, as seen in MCL1 transgenic mice and a variety of human malignancies. Our work progressed from the identification of MCL1 to studies of its effects and regulation, and is now unraveling a complex web of post-translational modifications. Our long-term goal is to delineate the mechanisms that regulate MCL1, and to understand how its dysregulation contributes to and thus might be targeted in cancer. The working hypothesis for our ongoing work is that MCL1 is subject to post-translational regulation through both exogenous signals and events linked to the cell cycle. Signaling-related regulation is illustrated by the ERK-inducible phosphorylation that slows the rapid turnover of the MCL1 protein. Cell cycle-related regulation is illustrated by the ERK-independent phosphorylation/band shift in G2/M phase which, along with an intriguing modification at the N-terminus (truncation), influences degradation as cells complete the cycle. AIM I will determine how signaling pathway-induced phosphorylation influences MCL1 stabilization/destabilization and thus its expression level and effects. This may maintain MCL1 expression in response to signals for viability- enhancement, or allow turnover for the induction of apoptosis. AIM II will dissect how the G2/M- phosphorylation/band shift and the N-terminal truncation control MCL1 degradation in cells traversing from one cycle to the next. This may aid in resetting MCL1 expression levels, and thus allow a fresh opportunity for regulation in newly divided cells. Our recent findings show that as transgenic mice grow older they demonstrate constitutive expression of MCL1 at levels normally seen upon stimulation. This is associated with increased resistance to cell death and occurs before tumors develop. It may thus allow the accumulation of further tumorigenic changes. Accordingly, AIM III will produce transgenic mice exhibiting expression of MCL1 at these increased levels, and/or containing forms of MCL1 that are less readily degraded through signaling- or cycle-related mechanisms. Elucidation of the multiple modes of MCL1 regulation, and the role of dysregulation in tumorigenesis, will suggest mechanisms that can be exploited and provide transgenic systems that can be utilized to target this BCL2 family member for the promotion of apoptosis at early as well as later stages of cancer.